PROJECT SUMMARY Cellular RNA levels in both healthy and diseased cells are highly dynamic, yet most analyses of RNA concentrations (RNA-sequencing, RNA-seq) capture only a static snapshot of cellular RNA. RNA levels are controlled at diverse steps in RNA metabolism including transcriptional initiation, transcriptional pausing, RNA processing, and RNA degradation. While RNA-seq provides a global method to identify RNAs that are up or down-regulated in response to biological perturbations, specialized experiments are required to determine which specific steps of RNA metabolism are affected. The specialized nature of these experiments, however, limits their wide-spread use. Thus, there is a pressing need to develop a flexible experimental platform that can be easily adapted to study the kinetics of a broad range of the regulated steps in RNA metabolism. The overall objective of this work is to add a temporal dimension to RNA- seq, transforming it from a static endpoint assay into a robust technique to measure the kinetics of RNA metabolism and reveal the diverse ways these kinetics are regulated and impacted by disease. This platform is based on metabolic labeling and improvements in nucleotide chemistry to study RNA dynamics at a range of timescales. The full potential of these approaches will be realized once they are integrated a robust and unified platform to examine RNA metabolism across a range of timescales (min to days), and transcript sizes (miRNA to long mRNA). Aim 1 is to determine optimal ways to globally distinguish changes in RNA synthesis from changes in RNA stability. The statistical power of these experiments will be systematically examined to define optimal experimental designs. Differences in RNA synthesis and degradation will be measured in cell lacking an enzyme that promotes mRNA decapping in comparison with wild type cells. The direct targets of this enzyme are expected to be post-transcriptionally stabilized but unchanged transcriptionally. Aim 2 will extend this system to measure the dynamics of short RNAs, including target directed miRNA degradation. Aim 3 includes the measurement the transient RNA expression and RNA processing, and the integration of these approaches to study the p53 tumor suppressor pathway. Successful completion of these aims will establish nucleotide recoding chemistry as a general platform to reveal RNA dynamics across distinct levels of gene expression.